The present invention relates generally to the fields of process for generating mammalian cell lines, especially human cell lines, and preferably human muscular cell lines, to cell lines produced by this process and to uses of these cell lines, especially in gene therapy.
Cell lines are widely used as in vitro models for studying the events involved during in vivo cellular or tissular development. For example, muscular development events can be reproduced during the differentiation of muscle cell lines. Accordingly, permanent mammalian cell lines, especially human cell lines, would be of considerable value for providing useful tools for dissecting the molecular and biochemical cellular events, for identifying and testing new drugs for mammalian diseases, such as dystrophies, for the study of myogenesis, etc . . .
The ability to establish particular cell lines in culture that continue to express properties characteristic of the cells in the tissue from which they were derived would have obvious advantages for studying effects of drugs and potentially toxic substances or for developping cellular gene therapy. Many, but not all, cell types have been propagated in culture, and some maintain their original characteristics although many lose their differentiated phenotype upon continuous passage in culture. The two major properties of cell lines are their capability to proliferate and to differentiate.
Many studies have shown that the action of multiple oncogenes is sufficient to convert primary cells into immortalized transformed cells. For example, Fogel et Defendi, 1967, PNAS, 58, 967-973 demonstrated that human myoblasts were susceptibles to infection with wild-type SV40 and that permanent cell lines could be generated following infection, however these rapidly lost the ability to differenciate.
Accordingly, the prior art is deficient in providing a satisfactory means for establishing mammalian, and especially human immortal cell lines. The present invention fulfills this longstanding need and desire in the art.
The present invention provides an improved process for establishing long-term mammalian cell line, from primary cells isolated from tissues biopsie or primary cell culture, especially originating from muscular biopsies obtained from Duchenne muscular dystrophy patients as well as from normal, dystrophin-positive individuals. The cell lines obtained according to the present invention show an great proliferative capacity and may prove valuable for in vitro investigations related to the cellular and molecular metabolisms, to new drug screening or to methods assessing for cellular toxicity or cellular damages.
Thus, the present invention first concerns a process for generating a mammalian cell line from primary mammalian cells, comprising the step of:
a) pre-treating a culture of said primary mammalian cells or a suspension thereof with at least one glucocorticoid,
b) optional step comprising obtaining a suspension of said pre-treated culture of step a),
c) transferring into the pre-treated cells of the suspension of step a) or b) at least one nucleic acid vector which is not of retroviral origin and which is competent to immortalize said pre-treated cells and
d) culturing the transferred cells of step c).